Characterization of reference genes for RT-qPCR in the desert moss Syntrichia caninervis in response to abiotic stress and desiccation/rehydration

Abstract

Syntrichia caninervis is the dominant bryophyte of the biological soil crusts found in the Gurbantunggut desert. The extreme desert environment is characterized by prolonged drought, temperature extremes, high radiation and frequent cycles of hydration and dehydration. S. caninervis is an ideal organism for the identification and characterization of genes related to abiotic stress tolerance. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) expression analysis is a powerful analytical technique that requires the use of stable reference genes. Using available S. caninervis transcriptome data, we selected 15 candidate reference genes and analyzed their relative expression stabilities in S. caninervis gametophores exposed to a range of abiotic stresses or a hydration-desiccation-rehydration cycle. The programs geNorm, NormFinder, and RefFinder were used to assess and rank the expression stability of the 15 candidate genes. The stability ranking results of reference genes under each specific experimental condition showed high consistency using different algorithms. For abiotic stress treatments, the combination of two genes (α-TUB2 and CDPK) were sufficient for accurate normalization. For the hydration-desiccation-rehydration process, the combination of two genes (α-TUB1 and CDPK) were sufficient for accurate normalization. 18S was among the least stable genes in all of the experimental sets and was unsuitable as reference gene in S. caninervis. This is the first systematic investigation and comparison of reference gene selection for RT-qPCR work in S. caninervis. This research will facilitate gene expression studies in S. caninervis, related moss species from the Syntrichia complex and other mosses.

Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Salix matsudana under different abiotic stresses

“…α-TUB2 encoding a cytoskeleton structure protein62 and DnaJ encoding a cellular chaperone have the ability to repair heat-induced protein machinery damage6364. Our results are in agreement with several previous studies, which showed that α-TUB2 and DnaJ were established as the most stable reference genes in plants under abiotic stresses; for example, in Syntrichia caninervis under drought, salt, and heavy metal stresses65, Corchorus capsularis under drought stress10, Buchloe dactyloides under salt stress66 , and Platycladus orientalis under salt stress67. Normalization with multiple reference genes is an effective way to avoid erroneous data that may result from using a single reference gene68.…”

“…Four reference genes, DnaJ, α-TUB2, MUB , and ACT under drought stress, were needed for accurate normalization. Two reference genes were found to be sufficient to analyze the expression of target genes in sorghum62, jute10, and moss65.…”

Carotenoids Overproduction in Dunaliella Sp.: Transcriptional Changes and New Insights through Lycopene β Cyclase Regulation

“…grown under high light stress [61]. Similarly, a second study realized with Syntrichia caninervis under abiotic stress; the α-Tub gene was also found to be stable amongst 15 reference genes, and 18S was the less unstable one [41].…”

“…Gene coding for Act, 18S RNA, α-TUB, and β-TUB was used for the screening of the most stable gene in all experiment conditions as they are known to be classic plant reference gene candidates [41]. Quantification cycle (Cq) values were imported into the comprehensive web-based tool RefFinder (http://150.216.56.64/referencegene.php?type=reference), which integrates commonly used reference gene evaluation programs together, including geNorm, NormFinder, Bestkeeper, and the comparative delta Ct methods [41,42]. Results elaborated by RefFinder were confirmed by GeNorm, which is available on excel file [43].…”

Suitable reference genes for real-time quantitative PCR in Salsola laricifilia under five abiotic stresses

“…However, these traditional reference genes are not always stably expressed under different conditions (Xiao et al 2015. Even for the same plant, reference genes may show different stability patterns under different stresses (Li et al 2015, Xiao et al 2015. Therefore, it is necessary to establish suitable reference genes for each species and tested them under certain experimental conditions in order to ensure their stability (Li et al 2012, Zhao et al 2016, Acevedo et al 2017.…”